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Table 7 Sample preparation procedure for LC-MS/MS analysis

From: A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice

  Procedure Notes
I. Reduction and alkylation of proteins
1. Add denaturing buffer to 50 μg protein of protein sample on ice (total volume should be 220 μL). · Denature protein.
· Can deal with samples at r.t. after denaturing protein samples.
·Should use low-protein-adsorption 1.5-mL tubes, e.g., SUMITOMO BAKELITE, SUMILON Proteosave SS 1.5 mL tubes, MS-4215 M.
· Do not pipet to prevent adsorption of proteins in pipette tips.
2. Add same amount of DTT as protein amount (Add 1 μL of 50 μg/μL DTT solution). · Do not pipet.
3. Stir the sample using a tube mixer (e.g., cute mixer CM-1000, EYELA) for 60 min at r.t. · Reduction of S-S bond.
4. Add 2.5-fold IAA of protein amount (Add 2.5 μL of 50 μg/μL IAA solution). · Do not pipet.
5. Stir the sample using a tube mixer for 60 min at r.t. in the dark. · Protection of –SH residue (alkylation)
· IAA can be degraded by light, so the sample tubes should be protected from light.
II. Methanol-chloroform precipitation (on ice)
6. Add 600 μL cold methanol to sample solution. Invert the tube. · Do not pipet.
7. Add 150 μL cold chloroform to sample solution. Invert the tube. · Do not pipet.
8. Immediately after adding 450 μL cold water to sample solution and inverting the tube, centrifuge the sample using swing rotor at 15,000 rpm for 5 min at 4°C. · Do not pipet.
9. Immediately after centrifugation, remove the upper layer (until a floating pellet). · The floating pellet is protein.
· Do not take the protein pellet.
10. Add 450 μL cold methanol to sample solution. Invert the tube gently to wash the protein pellet. · Do not pipet.
11. Centrifuge sample using swing rotor at 15,000 rpm for 5 min at 4°C.  
12. Immediately after centrifugation, remove the supernatant. · Do not take the protein pellet.
13. Again centrifuge sample using swing rotor at 15,000 rpm for 1 min at 4°C and remove the supernatant completely. · Do not take the protein pellet.
III. Double digestion with LysC and trypsin
14. Add 9 μL 6 M urea solution, and stir the sample using tube mixer for approximately 10 min at r.t. · Do not pipet.
15. Add 36 μL 0.1 M Tris–HCl buffer (pH 8.5). · Final concentration of urea is 1.2 M.
· Do not pipet.
16. Resuspend protein pellet by intermittent sonication with Branson 2510 sonicator. · Sonication for 30 seconds followed by a pause for 30 seconds on ice. Repeat this step until the pellet is resuspended.
17. Add 1/100-fold LysC of the protein amount (add 1 μL 0.5 μg/μL LysC solution). · Do not pipet.
· Gently tap using finger to stir sample solution.
18. Add 1% ProteaseMax solution (2.5 μL) so that the final concentration is 0.05%. · Do not pipet.
· Gently tap using finger to stir sample solution.
19. Incubate sample at 25°C for 3 h.  
20. Add 1/100-fold TPCK-trypsin of the protein amount (Add 1 μL of 0.5 μg/μL TPCK-trypsin solution). · Do not pipet.
· Gently tap using finger to stir sample solution.
21. Incubate sample at 37°C for 16 h. · Total volume is 49.5 μL.
IV. LC-MS/MS analysis
22. Add 7.5 μL of IS peptide mixture. · The concentration of the IS peptide mixture should be adjusted so that the injected amount of each peptide is 500 fmol for HPLC-API5000 or 100 fmol for HPLC-QTRAP5500.
· Pipet well in sample solution when adding IS peptide solution, then mix with vortex mixer.
23. Add 3 μL 50% formic acid in water. · Acidification.
· Mix with vortex mixer.
· Total volume is 60.0 μL.
24. Centrifuge sample at 15,000 rpm for 5 min at 4°C with an angular rotor.  
25. Apply 58 μL supernatant to 96-well plates or vials in autosampler. Keep the autosampler at less than 10°C.  
26. Inject 40 μL on LC-MS/MS. · 40 μL includes 33.3 μg peptide sample (50 μg protein × 40 μL/60 μL) and 100 fmol (QTRAP5500) or 500 fmol (API5000) of IS peptides.